On the analysis of long-chain alkane diols and glycerol ehters in biochemical studies

The chromatographic behavior of 1,2-, 1,3-, 1,4-, and 1,12-long-chain alkane diols and 1-O-alkylglycerols and their derivatives has been compared. Thin-layer chromatography on Silica Gel G gives poor separations of the 1,2-, 1,3-, and 1,4-alkane diols, O-alkylglycerols, and some of their isopropylidene derivatives. However, gas-liquid chromatography on 10% EGSS-X (coated on 100-120 mesh Gas-Chrom P) resolves the isopropylidenes of the alkane diols and O-alkylglycerols. We also document the formation of 1,3-alkane diols (after LiAlH(4) reduction) from 1-(14)C-labeled fatty acids incubated with mitochondrial fractions from heart and liver of rats. The labeled 1,3-alkane diol was identified by gas-liquid chromatography of its isopropylidene derivative and by its behavior after periodate oxidation. These results serve to caution investigators in the glycerol ether field against incorrect interpretation of data obtained on the incorporation of labeled fatty acids into alkyl ether bonds of glycerolipids. The methodology described points out a technique for distinguishing several types of alkane diols from O-alkylglycerols.     

Biphenyl dioxolanes as circular dichroism probes for the assignment of absolute configuration to aliphatic diols: Extending the scope to anti 1,n-diols and cyclic syn 1,2-diols

We describe herein the use of a flexible biphenyl moiety as efficient chirality probe in the assignment of the absolute configuration (AC) of aliphatic, non-chromophoric diols. The diols are transformed in the corresponding biphenyl dioxolanes in which the biphenyl system has either a P or M torsion depending on the chirality of the diol. As the correlation between biphenyl torsion and diol AC has been established and the sense of torsion is revealed by the sign of the biphenyl A band at 250 nm in the CD spectrum of the dioxolane, then the diols AC can be assigned simply looking at the CD spectra of these derivatives. This approach proved to be general, straightforward, and reliable for anti 1,2- 1,3-, and 1,4-diols bearing both one and two stereogenic centers and for cyclic syn 1,2-diols.     

Formation of cyclic products from the diepoxide of long-chain fatty esters by cytosolic epoxide hydrolase.

Since diepoxides are known metabolites of polyunsaturated fatty acids, the action of the cytosolic epoxide hydrolase purified from liver tissue was examined on these diepoxides. Diepoxymethylstearate was metabolized to the corresponding tetraol by high concentrations of affinity-purified cytosolic epoxide hydrolase. When the enzyme was diluted (1000- to 2000-fold), disappearance of the tetraol metabolite occurred simultaneously with formation of other hydration products with GC retention times and chromatographic mobilities different from those of the tetraol. The hydration products were identified as tetrahydrofuran diols based on comparison of chromatographic properties and mass spectral information with the properties and spectra of chemically generated products. Also, a mixture of diepoxymethylarachidonates was hydrated to tetraols using concentrated enzyme. As the enzyme was diluted (1000- to 2000-fold), a decrease in tetraol formation occurred along with the elevation of other hydration products whose mass spectra were consistent with tetrahydrofuran diol structures. These data are consistent with the epoxide hydrolase at low concentrations acting to open one epoxide followed by nonenzymatic cyclization to the tetrahydrofuran diols. The data also suggest that oxygenated lipids may be endogenous substrates for the cytosolic epoxide hydrolase. Since some oxylipins are known chemical mediators, the in vivo presence and role of these novel diols and tetrahydrofuran diols should be examined.     

The effect of manganese and ferric ions on the in vitro formation of dihydrodihydroxy metabolites of benzo(a)pyrene

The effect of ferric and manganese ions on the in vitro metabolism of benzo(a)pyrene (BP) to dihydrodihydroxy (diol) metabolites by rat liver microsomal preparations was studied. Of the 3 diols separated by high-pressure liquid chromatography (HPLC) and called diols 1, 2 and 3 in order of elution, diol 1 was identified by its U.V. spectrum as the 9,10-diol; diols 2 and 3 have not yet been identified positively but are probably the 4,5- and 7,8-diols respectively. Higher concentrations of both metals altered the diol profile; 10 and 50 mumol Fe3+ per incubation caused the disappearance of diols 1 and 2 and an increase in diol 3; 10 mumol Mn2+ caused a significant decrease in diol 2 while 50 mumol reduced diol 2 to a negligible amount and inhibited the formation of diol 1; both concentrations caused a relative increase in diol 3. If the tentative identification of diol 3 as the 7,8-diol is correct, manganese and ferric ions could be significant in the metabolism of BP to the active metabolite, the 7,8-diol-9,10-epoxide.     

Stereoselective biosynthesis of chloroarylpropane diols by the basidiomycete Bjerkandera adusta: exploring the roles of amino acids, pyruvate, glycerol and phenyl acetyl carbinol

Bjerkandera adusta produces many chlorometabolites including chlorinated anisyl metabolites (CAMs) and 1-arylpropane-1,2-diols (1, 2, 3, 4) as idiophasic metabolic products of L-phenylalanine. These diols are stereoselectively biosynthesized from a C7-unit (benzylic, from L-phenylalanine) and a C2-unit, of unknown origin, as predominantly erythro (1R,2S) enantiomers. Of the labeled amino acids tested as possible C2-units, at the 4-10 mM level, none were found to efficiently label the 2,3-propane carbons of the diols. However, glycine (2-13C), L-serine (2,3,3-d3) and L-methionine (methyl-d3) entered the biomethylation pathway. Neither pyruvate (2,3-13C2), acetate (1,2-13C2), acetaldehyde (d4) nor ethanol (ethyl-d5) labeled the 2,3-propane carbons of the diols at the 4-10 mM level. Pyruvate (2,3-13C2) and L-serine (2,3,3-d3) (which also entered the biomethylation pathway) did, however, effectively label the 2,3-propane carbons of the alpha-ketols and diols at the 40 mM level as evidenced by mass spectrometry. Glycerol (1,1,2,3,3-d5) also appeared to label one of the 2,3-propane carbons (ca. 5% as 2H2 in the C3 side chain) as suggested by mass spectrometric data and also entered the biomethylation pathway, likely via amino acid synthesis. Glycerol (through pyruvate), therefore, likely supplies C2 and C3 of the propane side chain with arylpropane diol biosynthesis. Incubation of B. adusta with synthetic [2-2H1, 2-18O]-glycerol showed that neither 2H nor 18O were incorporated in the alpha-ketols or diols. The oxygen atom on the C2 of the ketols/diols, therefore, does not appear to come from the oxygen atom on the C2 of glycerol. Glycerol, however, can readily form L-serine (which can then form pyruvate via PLP/serine dehydratase and involve transamination washing out the 18O label and providing the oxygen from water), and can then go on to label the C2-unit. Labeled alpha-ketol, phenyl acetyl carbinol (5) (PAC; ring-d(5), 2,3-13C2 propane) cultured with B. adusta leads to stereospecific reduction to the (1R,2S)-diol (6) (ring-d5 and 2,3-13C2); in all other metabolites produced, the 2,3-13C2) label is washed out. Incubation of the fungus with 4-fluorobenzaldehyde (13) produces a pooling of predominantly erythro (1R,2S) 1-(4'-fluorophenyl)-1,2-propane diol (18 as diacetate) (through the corresponding alpha-ketols 16, 17). Blocking the para-position with fluorine thus appears to prevent ring oxygenation and also chlorination, forcing the conclusion that para-ring oxygenation precedes meta-chlorination.     

Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases

Linoleic acid diol glucuronides have been isolated previously from urine of patients suffering from generalized peroxisomal disorders. Glucuronidation of linoleic acid and linoleic acid diols by human liver microsomes was studied to investigate the role of glucuronide conjugation in the metabolism of linoleic acid diols. Glucuronide products were isolated and analyzed by TLC and HPLC-MS. HPLC-MS showed ions with (m/z) corresponding to singly glucuronidated linoleic acid diols while TLC revealed that the glucuronidation was at a hydroxyl position. Kinetic analysis gave apparent K(m) values in the range of 50-200 microM and V(max) rates from 5 to 12 nmol/mg x min. These rates are substantially higher than activities seen for most endogenous hydroxylated substrates. Assays using each of the four individually purified linoleic acid diol enantiomers suggest that glucuronidation occurs at only one of the two hydroxyl groups of each enantiomer. These results show for the first time that hydroxylated fatty acids are actively glucuronidated by human liver microsomes and suggest that glucuronidation may play a significant role in the biotransformation of linoleic acid diols in humans.     

An approach based on liquid chromatography/electrospray ionization-mass spectrometry to detect diol metabolites as biomarkers of exposure to styrene and 1,3-butadiene

Styrene and 1,3-butadiene are important intermediates used extensively in the plastics industry. They are metabolized mainly through cytochrome P450-mediated oxidation to the corresponding epoxides, which are subsequently converted to diols by epoxide hydrolase or through spontaneous hydration. The resulting styrene glycol and 3-butene-1,2-diol have been suggested as biomarkers of exposure to styrene and 1,3-butadiene, respectively. Unfortunately, poor ionization of the diols within electrospray mass spectrometers becomes an obstacle to the detection of the two diols by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). We developed an LC/ESI-MS approach to analyze styrene glycol and 3-butene-1,2-diol by means of derivatization with 2-bromopyridine-5-boronic acid (BPBA), which not only dramatically increases the sensitivity of diol detection but also facilitates the identification of the diols. The analytical approach developed was simple, quick, and convincing without the need for complicated chemical derivatization. To evaluate the feasibility of BPBA as a derivatizing reagent of diols, we investigated the impact of diol configuration on the affinity of a selection of diols to BPBA using the established LC/ESI-MS approach. We found that both cis and trans diols can be derivatized by BPBA. In conclusion, BPBA may be used as a general derivatizing reagent for the detection of vicinal diols by LC/MS.     

Substrate and inhibitor selectivity,and biological activity of an epoxide hydrolase from Trichoderma reesei

Molecular Biology Reports - Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the...     

The effects of certain glycols, substituted glycols and related organic solvents on the thermal stability of soluble collagen

The effects of a number of related diols, substituted diols and glycerol on the thermal stability of acid-soluble calf skin collagen were investigated. Thermal transition temperatures were determined by optical rotation measurement. Short-chain diols with terminal hydroxyl groups, i.e. ethylene glycol and propane-1,3-diol, stabilized the protein at all accessible concentrations. Stabilization was also observed with glycerol and diethylene glycol. Higher homologues in the diol series produced various effects, as did hydroxyl-group positional isomerism. Monoalkyl substitution of diols progressively lowered the denaturation temperature of collagen. Results are discussed in relation to possible mechanisms of perturbant action.     

Enzymic Deacetylation of Methyl 2,3-Di-O-Acetyl-α- and β-D-Threofuranoside

Enzymic deacetylation of methyl 2,3-di-O-acaetyl-α- and β-D-threofuranoside using porcine liver esterase affords monoacetates only (in the case of the α-anomer, the 3-O-acetylderivative exclusively) without any further hydrolysis to the corresponding diols.     

Structure and absolute configuration of new acetylenic compounds isolated from cultures of Clitocybe catinus

Investigations of the extracts of a culture of Clitocybe catinus gave rise to the isolation of new acetylenic diols 1-3. Their structure was determined on the basis of 1H- and 13C-NMR evidence and the absolute configuration elucidated by means of the modified Mosher's method.     

α-Carbonic anhydrases are sulfatases with cyclic diol monosulfate esters

Carbonic anhydrases (CA) catalyze activated ester hydrolysis in addition to the hydration of CO(2) to bicarbonate. They also show phosphatase activity with 4-nitrophenyl phosphate as substrate but not sulfatase with the corresponding sulfate. Here we prove that the enzyme is catalyzing the synthesis of cyclic diols from sulfate esters. 5-, 6- and 8-membered ring cyclic sulfates incorporating a neighboring secondary alcohol moiety were treated with CA II and yielded the corresponding cyclic diols. Inhibitory properties of obtained cyclic and original sulfate esters were then investigated on human carbonic anhydrase I (hCA I), hCA II, hCA IV and hCA VI (h?=?human isoform). K(I)-s of these compounds ranged between 32.7-423 μM against hCA I, 2.13-32.4 μM against hCA II, 13.7-234 μM against hCA IV and 76-278 μM against CA VI, respectively. The sulfatase activity of CA with such esters is amazing considering the fact that 4-nitrophenyl-sulfate is not a substrate of these enzymes.     

Partition behavior of hydrophilic diols in an ethanol/ammonium sulfate salting‐out extraction system

It is important to investigate the partition behavior of hydrophilic alcohols at different process parameters in order to have a deep understanding of the salting‐out extraction technique and to design suitable downstream processes. First, phase diagram data of ethanol/ammonium sulfate at 293.15 K were obtained, and the reliability of binodal curve and tie line data were proved by Merchuk, Othmer–Tobias, and Bancroft equations, respectively. Then, the partition behavior of five short‐chain diols was studied. For different tie lines, the partition coefficient changed linearly with an increase in tie line length. The concentration of diols also had a significant effect on the partition behavior due to the similarity between diols and ethanol. Further, the effect of temperature was increased as the hydrophobicity of the diols increased, and the partition behavior of diols was correlated with their hydrophobicity, implying that the solutes with higher hydrophobicity could be extracted more effectively. These findings are useful for designing an economic and efficient salting‐out extraction process for diverse products.     

Oxidation of 17alpha,20beta- and 17alpha,20alpha-dihydroxysipregn-4-en-ones--side products of biotransformation of progesterone by recombinant microorganisms expressing cytochrome P45017alpha

Progesterone biotransformation with recombinant yeast Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 alpha expressing bovine adrenocortical cytochrome P45017 alpha yielded 17 alpha-hydroxyprogesterone and two diols, 17 alpha, 20 beta- and 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17-20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20 beta-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17 alpha-hydroxylation and 20 alpha- and 20 beta-reduction. The results widen the possibilities for enzymatic and chemical modifications of steroids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Identification and quantitation of lipid-bound short-chain diols

Procedures are described for the hydrolysis of neutral lipid fractions containing long-chain esters and alk-1-enyl ethers of short-chain diols, and for the identification and quantitation of the constituent diols as long-chain cyclic acetals using gas-liquid chromatography in combination with mass spectrometry.     

Urinary excretion of diols derived from eicosapentaenoic acid during n-3 fatty acid ingestion by man.

Epoxides and fatty acid diols derived from arachidonate by the action of cytochrome P-450 appear in human urine and have biological activities. Dietary eicosapentaenoic acid gives rise to prostaglandins in vivo, but vascular effects of n-3 supplements do not all correlate with altered types or amounts of in vivo cyclooxygenase products. We investigated whether dietary eicosapentaenoic acid could also be metabolized by cytochrome P-450, by assessing the excretion of its vicinal diols. Utilizing gas chromatography/negative chemical ionization mass spectrometry, we have found that humans ingesting n-3 fatty acids excrete vicinal diols of eicosapentaenoic acid in substantial quantities.     

Alcohols produce reversible and irreversible acceleration of phospholipid flip-flop in the human erythrocyte membrane.

The slow, non-mediated transmembrane movement of the lipid probes lysophosphatidylcholine, NBD-phosphatidylcholine and NBD-phosphatidylserine in human erythrocytes becomes highly enhanced in the presence of 1-alkanols (C2-C8) and 1,2-alkane diols (C4-C8). Above a threshold concentration characteristic for each alcohol, flip rates increase exponentially with the alcohol concentration. The equieffective concentrations of the alcohols decrease about 3-fold per methylene added. All 1-alkanols studied are equieffective at comparable calculated membrane concentrations. This is also observed or the 1,2-alkane diols, albeit at a 5-fold lower membrane concentration. At low alcohol concentrations, flip enhancement is reversible to a major extent upon removal of the alcohol. In contrast, a residual irreversible flip acceleration is observed following removal of the alcohol after a treatment at higher concentrations. The threshold concentrations to produce irreversible flip acceleration by 1-alkanols and 1,2-alkane diols are 1.5- and 3-fold higher than those for flip acceleration in the presence of the corresponding alcohols. A causal role in reversible flip-acceleration of a global increase of membrane fluidity or membrane polarity seems to be unlikely. Alcohols may act by increasing the probability of formation of transient structural defects in the hydrophobic barrier that already occur in the native membrane. Membrane defects responsible for irreversible flip-acceleration may result from alterations of membrane skeletal proteins by alcohols.     

Synthesis of epoxide and vicinal diol regioisomers from docosahexaenoate methyl esters

Docosahexaenoic acid (22:6(n-3)) was recently shown to be metabolized by liver microsomes to vicinal diol regioisomers. To identify the diols, and to compare their biological actions with those of epoxide precursors, we developed a chemical method to synthesize microgram to milligram amounts of epoxides and corresponding diols. In brief, methylated docosahexaenoate was reacted for 15 min with 0.1 eq m-chloroperoxybenzoic acid. After normal- and reverse-phase high performance liquid chromatography, six products were isolated. The underivatized or hydrogenated products were characterized and identified using capillary gas-liquid chromatography and mass spectrometry. The products were identified as 19,20-, 16,17-, 13,14-, 10,11-, 7,8-, and 4,5-epoxy-docosapentaenoate. Per incubation, the total epoxide yield from 22:6(n-3) was 8.6%. By reincubating unused substrate 10-20 times (cycling), the total epoxide could be increased to 55-70%. As found for epoxides of arachidonic and eicosapentaenoic acids, the yield of individual regioisomers increased as the distance between the targeted double bond and carbomethoxy group increased. Each epoxide regioisomer was hydrolyzed to its corresponding vicinal diol. The gas-liquid chromatographic retention times and mass spectra of the diol products were found to match those of metabolites produced by cytochrome P-450 monooxygenases.     

聚乳酸基热固性聚氨酯及其形状记忆性能

通过聚乳酸二元醇和聚乳酸-聚己内酯共聚物二元醇与六亚甲基二异氰酸酯(HDI)三聚体交联反应合成了一系列生物基热固性聚氨酯(Bio-PUs)。利用傅里叶红外(FTIR)、差示扫描量热分析(DSC)、热失重分析(TGA)、万能拉伸机和细胞毒性等测试方法对获得的聚乳酸基聚氨酯进行了表征。结果表明,与聚乳酸二元醇相比,聚乳酸-聚己内酯共聚物二元醇降低了生物基热固性聚氨酯的玻璃化温度(Tg),提高了热固性聚氨酯的热稳定性;且聚乳酸-聚己内酯型聚氨酯的力学性能和形状记忆性能更为优异。其中,聚乳酸-聚己内酯共聚物二元醇分子量为3 000时得到的热固性聚氨酯(Bio-PU2-3000)的杨氏模量为277.7 MPa,伸长率为230%;聚乳酸-聚己内酯共聚物二元醇分子量为1 000得到的热固性聚氨酯(Bio-PU2-1000)在人体体温下的形变回复时间仅为93 s。另外,通过显微镜观察到细胞在含聚乳酸基热固性聚氨酯的培养液中生长状态良好,表明制备得到的生物基聚氨酯无细胞毒性。     

Molecular engineering of epoxide hydrolase and its application to asymmetric and enantioconvergent hydrolysis

Safety and regulatory issues favor increasing use of enantiopure compounds in pharmaceuticals. Enantiopure epoxides and diols are valuable intermediates in organic synthesis for the production of optically active pharmaceuticals. Enantiopure epoxide can be prepared using epoxide hydrolase (EH)-catalyzed asymmetric hydrolysis of its racemate. Enantioconvergent hydrolysis of racemic epoxides by EHs possessing complementary enantioselectivity and regioselectivity can lead to the formation of enantiopure vicinal diols with high yield. EHs are cofactor-independent and easy-to-use catalysts. EHs will attract much attention as commercial biocatalysts for the preparation of enantiopure epoxides and diols. In this paper, recent progress in molecular engineering of EHs is reviewed. Some examples and prospects of asymmetric and enantioconvergent hydrolysis reactions are discussed as supplements to molecular engineering to improve EH performance.